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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 7 2329-2336
Copyright © 1997 by The Endocrine Society

Experimental Studies

Effect of Interferon-{gamma} and Glucose on Major Histocompatibility Complex Class I and Class II Expression by Pancreatic ß- and Non-ß-Cells1

Dejan Pavlovic, Marnix Van De Winkel, Bart Van der Auwera, Meng-Chi Chen, Frans Schuit, Luc Bouwens and Daniel Pipeleers

Diabetes Research Center, Vrije Universiteit Brussel, 1090 Brussels, Belgium

Address all correspondence and requests for reprints to: Prof. D. Pipeleers, Department of Metabolism and Endocrinology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Surface major histocompatibility complex (MHC) class I and class II expression by pancreatic islet cells is considered a local initiator or regulator of immune processes that can lead to diabetes. Locally released cytokines, in particular interferon-{gamma}, are known to stimulate MHC antigen expression by islet cells. The present study quantifies MHC expression in cultured pancreatic ß- and non-ß-cells from both rat and human organs. Interferon-{gamma} increased MHC class I expression in endocrine ß- and non-ß-cells as well as in pancreatic ductal cells. The cytokine induced a 6-fold increase in the MHC class I messenger ribonucleic acid levels in pancreatic ß-cells; this effect was 2-fold amplified in the presence of elevated glucose levels (20 mmol/L instead of 6 mmol/L). No MHC class II expression was observed in endocrine ß- or non-ß-cells; human, but not rat, ductal cells exhibited MHC class II expression that increased in the presence of interferon-{gamma}.

These data indicate that the increase in ß-cell MHC class I expression described in the pancreata of diabetic patients may result from stimulated transcription after exposure to locally released interferon-{gamma} and/or to a hyperglycemic state. The association of human islets with ductal cells in which MHC class II expression is stimulated by interferon-{gamma} makes these cells potential participants in the autoimmune process in diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SURFACE expression of major histocompatibility complex (MHC) antigens by nonimmune cells can regulate their interactions with immune cells (1, 2, 3, 4, 5). In the endocrine pancreas, hyperexpression of MHC class I antigens and aberrant expression of MHC class II antigens have been suggested to induce lymphocyte infiltrations and subsequent local reactions (6, 7, 8, 9, 10, 11, 12, 13). The products of immune cells, cytokines, were shown to stimulate MHC class I and/or class II expression in the islets of Langerhans (14, 15, 16, 17). Although this general concept has been supported by in vitro and in vivo studies in both rodents and humans, the specific data are sometimes controversial, raising questions about the cell, strain, and species specificity of the observed phenomena (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). The complexity of the studied models in terms of both interactive agents and cell composition makes it difficult to clearly identify the events at the level of the ß-cells. For these reasons, we have compared MHC class I and class II expression on the surface of pancreatic ß- and non-ß-cells that were isolated from normal rat or human organs. We choose to examine the effect of interferon-{gamma} because this cytokine is known for its stimulatory action on MHC expression (14, 15, 16, 17), it has been demonstrated in mononuclear cell infiltrates of diabetic islets (10, 12, 13), and treatment with anti-interferon-{gamma} antibodies suppresses the inflammatory reactions and the MHC class I hyperexpression in mice (18, 19). The cell preparations were studied at basal and high glucose levels because glucose-induced activation of ß-cells may facilitate antigen expression by these cells (20, 21).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation

Rat pancreases were obtained from 10-week-old male Wistar rats (Heverlee colony, Belgium). Pancreases were digested with collagenase and filtered over 500-µm pore size mesh nylon screens. Filtered material was dissociated and purified into single cell preparations containing either more than 90% ß-cells or more than 85% endocrine non-ß cells (22). Pancreatic ductal cells were purified from the tissue fragments that remained on the filters, using centrifugation on an isoosmotic 1.06 g/mL Percoll density cushion (23). Rat splenocytes were isolated by Lymphoprep according to the manufacturer’s instructions (Nycomed Pharma, Oslo, Norway).

Human pancreatic endocrine and nonendocrine cells were isolated from seven pancreata that were obtained from adult heart-beating organ donors and processed at the Central Unit of the ß Cell Transplant (Brussels, Belgium), as previously described (24, 25). Collagenase digests were separated by Ficoll gradient purification into islet and nonislet fractions. Nonislet fractions were cultured for a minimum of 3 days at 37 C to obtain ductal cells at greater than 90% purity. The mean donor age was 38 \ 5 yr (mean \ SEM; range, 17–58 yr). For messenger ribonucleic acid (mRNA) analysis, islet cell preparations were used with more than 75% insulin-positive cells. Human white blood cells were isolated from whole blood by the FACS lysing solution according to the manufacturer’s instructions (Becton Dickinson, Sunnyvale, CA).

Islet cell cultures

The rat purified islet cell preparations were first reaggregated by 3 h of orbital shaking in a rotary CO2 incubator (Braun, Melsungen, Germany) and then cultured for 16 h at 37 C in 5% CO2 before starting the experiments. Human islet cell preparations were used after culture for 4–17 days at 37 C in 5% CO2. Ham’s F-10 medium with 6 or 20 mmol/L glucose and 50 µmol/L 3-isobutyl-1-methylxanthine was used for cultures at 37 C, and CO2-independent medium with 10 mmol/L glucose was used for incubations at 20 C (Life Technologies, Paisley, Scotland). Media were supplemented with 0.5% BSA (Boehringer Mannheim, Mannheim, Germany), 0.1 mg/mL streptomycin (Continental Pharma, Puteaux, Belgium), 125 U/mL penicillin (Laboratoires Diamant, Brussels, Belgium), and 2 mmol/L L-glutamine (Life Technologies). Cultures of aggregated cells were carried out in suspension dishes (Nunc, Naperville, IL), 5 x 104 to 5 x 105 cells in 3 mL medium (3-cm dish) and 2 x 106 to 3 x 106 cells in 9 mL medium (9-cm dish). The cultures of single cells were carried out in slide chambers (Nunc), 5–10 x 103 cells/cup in 0.5 mL medium. The effect of interferon-{gamma} was assessed in rat cells after exposure to the recombinant murine form (100 U/mL, 98% pure, 10 U/ng; Holland Biotechnology, Leiden, The Netherlands) and in human cells cultured with the recombinant human form (1000 U/mL, 97% pure, 47, 5 U/ng; Genzyme, Cambridge, MA). At the end of these incubations, islet cell aggregates were dissociated in calcium-free medium to which trypsin (50–100 µg/mL) and deoxyribonuclease were added for preparations that were cultured at 37 C (22, 25).

Immunocytochemistry

Cell preparations were suspended in phosphate-buffered saline containing 0.5% BSA for studies on surface expression of MHC class I and class II antigens. Samples of 5 x 104 cells/100 µL were incubated with the anti-class I or class II antibodies for 60 min at room temperature, washed, and then further incubated for 30 min with a phycoerythrin- or 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-conjugated second antibody. For intracellular stainings, cells were first fixed in 2% (vol/vol) formaldehyde and permeabilized by 10-min incubation with Triton X-100 \[0.1% (vol/vol) in phosphate-buffered saline-0.5% BSA\]. They were than incubated with antibodies to insulin (30 min at room temperature) or ductal cell marker cytokeratin-19 (26, 27) (overnight at 4 C), washed, and then exposed to a second antibody for an additional 30 min. Stainings on paraffin sections were carried out as previously described (28). The following antibodies were used: mouse MRC OX-18 antibody to a monomorphic determinant of rat class I MHC antigen (RT1.A), mouse MRC OX-6 antibody to a monomorphic determinant of the rat class II MHC antigen (RT1.B, Serotec, Oxford, UK), phycoerythrin-conjugated donkey anti-mouse antibody, phycoerythrin- and AMCA-conjugated goat anti-rabbit and donkey anti-rat antibodies (Jackson Laboratories, West Grove, PA), fluorescein-conjugated goat anti-mouse antibody (Southern Biotechnology Associates, Birmingham, AL), sheep anti-insulin antibody conjugated with fluorescein (Serotec), rabbit antihuman ß2-microglobulin antibody (Zymed, San Francisco, CA), mouse CR3/43 antibody to HLA-DR (Dako, Glostrup, Denmark), rat antibody YAML55.6 to a monomorphic determinant of the human class II antigen (Serotec), and mouse antibody RCK108 to cytokeratin CK-19 (Dako). Rat splenocytes and human lymphocytes were added as positive controls for MHC class I and class II expression. Negative controls were prepared either by omission of the first antibody or by use of isotype-matched control antibodies or appropriate control serum.

Cellular analysis

Antibody binding to cells was detected by light or fluorescence microscopy. For quantitative analysis, dissociated cells were examined in a FACStar flow cytometer (Becton Dickinson, Sunnyvale, CA) equipped with two lasers, one emitting UV light for excitation of AMCA (a water-cooled ion laser Innova 90 from Coherent, Palo Alto, CA) and one emitting a 488-nm monochromatic light beam for excitation of phycoerythrin and fluorescein (an air-cooled argon laser ILT model 5500ASL from ILT, Salt Lake City, UT). The gates for analysis were selected on forward vs. side scatter after running a propidium iodide-treated control sample of unfixed cells, which allowed definition of the windows for living and dead cells and cell debris. Approximately 104 cells/sample were analyzed.

Competitive reverse transcriptase-PCR (RT-PCR)

The competitive RT-PCR was performed by modification of a previously described technique that involved coamplification of mRNA after reverse transcription with an artificial internal standard (mimic) that differs in length from the target (29). mRNA was isolated from cultured cell aggregates using oligo(deoxythymidine)25-coated polystyrene Dynabeads (Dynal, Oslo, Norway). The reverse transcription mixture was prepared with the GeneAmp RNA PCR Kit (Perkin-Elmer, Norwalk, CT). The reaction solution was composed of 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L deoxy-NTP, 2.5 µmol/L random hexamer primer, 1 U/µL ribonuclease inhibitor, and 2.5 U/µL Moloney murine leukemia virus reverse transcriptase. It was first incubated at room temperature for 10 min, then at 42 C for 1 h, and finally heated at 99 C for 5 min before cooling on ice.

The two competitor DNAs, MRT1A and MHLA1, serving as internal standards (mimics) for rat RT1.A and human HLA-A, -B, and -C complementary DNAs (cDNAs) were constructed using a PCR MIMIC Construction Kit (Clontech, Palo Alto, CA). Neutral DNA, a BamHI/EcoRI fragment of the v-erbB oncogene, was first amplified with a composite primer pair, each composed of a target gene-specific sequence linked to a sequence that anneals to the neutral DNA template. Composite DNA primer pair sequences are MRT1A-F (5'-GCTCACACTCGCTGCGGTATCAAGTTTCGTGAGCTGATTG-3') and MRT1A-R (5'-GCCATACATCTCCTGGATGGTGAGTCCATGGGGAGCTTT-3'), and MHLA1-F (5'-AGTGGGCTACGTGGACGACACAAGTTTCGTGAGCTGATTG-3') and MHLA1-R (5'-ATGTAATCCTTGCCGTCGTATTGAGTCCATG-GGGAGCTTT-3'; sequences that anneal to neutral DNA template are italicized). The resulting fragments were then diluted 1:100 and reamplified with target-specific primer pairs for rat RT1A gene RT1A-F (5'-GCTCACACTCGCTGCGGTAT-3') and RT1A-R (5'-GCCATACATCTCCTGGATGG-3'), and for HLA-A, -B and -C genes HLA1-F (5'-AGTGGGCTACGTGGACGACA-3') and HLA1-R (5'-ATGTAATCCTTGCCGTCGTA-3'). The prepared competitor DNA contains the same primer annealing sequences as the target cDNA, but its PCR product is longer (476 vs. 299 bp). It was purified by passage through a Chroma SpinTE-100 Column (Clontech), monitored spectrophotometrically at 260 and 280 nm, and than diluted in 50 mg/mL glycogen.

In competitive PCR experiments, decreasing amounts of competitor DNA were added to the PCR reaction mixtures containing a constant amount of cDNA sample. PCR reactions were carried out in a TC 9600 thermocycler (Perkin-Elmer/Cetus, Norwalk, CT). The PCR mixture contained 5 µL cDNA prepared in the reverse transcription, 2 µL competitor DNA, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2 mmol/L MgCl2, 0.4 µmol/L target-specific primers, 0.2 mmol/L of each deoxy-NTP, and 0.625 U AmpliTaq DNA polymerase (Perkin-Elmer/Cetus) in a final volume of 25 µL. PCR conditions were as follows: 35 cycles, each consisting of denaturation for 45 s at 94 C, annealing for 45 s at 58 C for the rat RT1.A gene primer pair and 61 C for human HLA-A, -B, and -C genes primer pair, and extension for 90 s at 72 C, preceded by an initial denaturation of 2.5 min at 94 C. The last PCR step was a 10-min extension at 72 C.

Five microliters of PCR products were separated by electrophoresis in 2% MetaPhore agarose gel (FMC Bioproducts, Rockland, ME). The ethidium bromide-stained gels were photographed under UV transillumination using Polaroid type 665 film (Cambridge, MA). The fragment intensities of the negative films were scanned by an Enhanced Laser Densitometer Ultroscan XL (LKB, Bromma, Sweden) and expressed in arbitrary units.

Statistical data analysis

Statistical analysis of the data was performed by StatView 512 (BrainPower, Calabalas, CA). Data was compared by ANOVA and paired Student’s t test; the significance of the differences between various conditions was determined by Fisher’s protected least significant difference and Scheffe’s F tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Microscopical analysis

Rat pancreatic ß-cells exhibited a fluorescent membrane staining for MHC class I antigens. After 6 days of culture at 37 C with 6 mmol/L glucose, more than 95% of the cells were positive; this percentage as well as the cellular fluorescence intensities were higher than shortly after isolation or after culture at 20 C (60–80% positive cells). The fluorescence intensity for MHC class I antigens was increased by the presence of interferon-{gamma}. In none of these preparations were ß-cells detected with membrane staining for MHC class II antigens. Rat pancreatic endocrine non-ß-cells and ductal cells also expressed MHC class I antigens and were negative for MHC class II. Interferon-{gamma} increased MHC class I expression, but did not induce MHC class II in these cell preparations.

After 6 days of culture, human insulin-positive cells exhibited a surface expression of ß2-microglobulin (Fig. 1Go, A and A') which was up-regulated by exposure to interferon-{gamma} (Fig. 1Go, B and B'). They were negative for MHC class II regardless of whether interferon-{gamma} was present (Fig. 1Go, C and C'). The interferon-{gamma}-exposed preparations, however, contained a number of insulin-negative cells that were MHC class II positive (Fig. 1Go, C and C'). When human ductal cell aggregates were examined, 5–10% of the cells were positive for MHC class II antigens; interferon-{gamma} increased this percentage to over 60%. As virtually all cells in this preparation were positive for the cytokeratin CK-19 ductal cell marker, it can be concluded that the MHC class II-positive cells correspond to ductal cells (Fig. 2Go). These cells also exhibited a surface expression of ß2-microglobulin that was up-regulated by exposure to interferon-{gamma}.



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  		Figure 1. Human pancreatic cells after double staining with fluorescein for insulin (A, B, and C), and phycoerythrin for membranous ß2-microglobulin (A' and B') or HLA-DR (C'). The cells were first cultured for 6 days at 37 C with 10 mmol/L glucose without (A and A') or with (B, B', C, and C') human interferon-{gamma} (1000 U/mL). The presence of interferon-{gamma} increased membrane staining for ß2-microglobulin on insulin-positive cells (B and B'), but did not induce membrane staining for HLA-DR (C and C'). Insulin-negative cells were found with positivity for HLA-DR (C and C'). Magnification, x540.

 


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  		Figure 2. Immunocytochemical staining on consecutive paraffin sections of human ductal cell preparations that were cultured for 6 days without (A and A') or with interferon-{gamma} (B and B'). Cells were stained for HLA-DR (A and B) and the ductal marker CK-19 (A' and B'). Magnification, x200.

 
Flow cytometric analysis

Cellular fluorescence intensities were compared by flow cytometry. Staining of rat ß-cells with MHC class I antibodies was lowest after 16 h of culture at 37 C or after 6 days of culture at 20 C; it was 2-fold higher after 6 days of culture at 37 C, with similar values at 6 or 20 mmol/L glucose (Fig. 3Go and Table 1Go). Culture with interferon-{gamma} resulted in 3-fold higher fluorescence intensities (Fig. 3Go and Table 1Go). This effect was similar at 6 and 20 mmol/L glucose (not shown). After MHC class II staining, less than 5% positive cells were detected; these cells exhibited a diffuse cytoplasmic fluorescence, which is typical for damaged cells. Control experiments indicated that exposure to trypsin did not affect MHC class I and class II staining in splenocytes, nor did it impair MHC class I staining in single ß-cells (data not shown).



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  		Figure 3. Effects of different culture conditions on surface MHC class I and class II expression by rat ß-cells. After culture for 6 days, rat ß-cell aggregates were dissociated, and isolated cells were analyzed for their surface antigens by flow cytometry. Splenocytes and ß-cells exposed to the isotype-matched control antibody served as negative controls; splenocytes stained for MHC class I and class II were used as positive controls.

 

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  		Table 1. Effects of temperature, glucose concentration, and IFN-{gamma} on MHC class I expression by rat pancreatic ß-cells

 
Flow cytometry of human islet cell preparations showed a 3- to 4-fold increase in ß2-microglobulin expression after interferon-{gamma} exposure, whereas no effect on MHC class II expression was observed (Fig. 4Go). Pancreatic ductal cells were also positive for ß2-microglobulin, with a fluorescence intensity that increased 2- to 3-fold after culture with interferon-{gamma} (not shown). Double labeling for MHC class II and CK-19 ductal cell marker indicated that this pancreatic cell preparation contains MHC class II-positive cells, particularly after culture with interferon-{gamma} (Fig. 5Go).



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  		Figure 4. Effect of culture with interferon-{gamma} on surface ß2-microglobulin and HLA class 2 expression by human islet cells. After culture for 6 days, human islets were dissociated, and isolated cells were analyzed for their ß2-microglobulin and HLA class 2 surface expression. Gates were selected by forward scatter and fluorescence intensity on the population of islet cells that was fluorescently labeled with an insulin antibody.

 


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  		Figure 5. Effect of culture with interferon-{gamma} on HLA class II expression by human ductal cells. After 6 days of culture, human ductal cell aggregates were dispersed, and cells were double stained for HLA class II (AMCA fluorescence) and CK-19 (FITC fluorescence). Flow cytometry was used to detect the presence of double positive cells (window B). These experiments made use of anti-HLA class II antibodies, because the anti-HLA-DR antibodies used in Fig. 2Go were not suitable for double staining given their common source (mouse).

 
Competitive RT-PCR

The cellular content in mRNA for MHC class I was quantified by competitive RT-PCR (Fig. 6Go). In purified rat ß-cells, the content in RT1.A mRNA was 3-fold higher after 6 days of culture with 20 mmol/L glucose than after culture with 6 mmol/L glucose (Table 2Go). Independently of the glucose effect, interferon-{gamma} increased RT1.A mRNA expression approximately 5-fold (Table 2Go). Similar results were obtained with human islet cell preparations. The HLA-A, -B, and -C mRNA was 2-fold higher at 20 mmol/L glucose than at 6 mmol/L, and addition of interferon-{gamma} increased these levels 6-fold at both glucose concentrations (Table 2Go).



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  		Figure 6. Representative example of competitive RT-PCR showing the effect of interferon-{gamma} on RT1.A gene expression by rat pancreatic ß-cells cultured with 6 mmol/L glucose. Each PCR reaction was carried out on a cDNA equivalent for 5 x 103 cells. Lanes 1–5 and 7–11 contained decreasing amounts of competitor DNA, respectively 8, 2, 0.5, 0.125, and 0.031 attomoles. PCR products of RT1.A cDNA (229 bp) and competitor DNA (476 bp) were separated by 2% agarose gel electrophoresis. Lane 6 contains HaeIII-digested pBR322 as a DNA size marker.

 

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  		Table 2. Effects of glucose and IFN{gamma} on MHC class I mRNA levels in pancreatic islet cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Microscopic analysis of tissue sections has indicated that pancreatic ß-cells exhibit an increased expression of MHC class I antigens at the onset of autoimmune diabetes (6, 7, 8, 9, 11). In vitro experiments on isolated islets have suggested that locally released cytokines might be responsible for this phenomenon (14, 15, 16, 17). Several observations favor a role of interferon-{gamma} in this process. This cytokine is known for its stimulating action on MHC class I expression in various cell types (3, 14, 15, 16, 17, 30, 31, 32). It has also been associated with the development of the insulitis process (10, 12, 13, 18, 33). The present experiments on purified rat ß-cells demonstrate that interferon-{gamma} interacts directly with these cells, increasing their MHC class I expression at both the mRNA and cell surface levels. These effects were quantified by, respectively, competitive RT-PCR and flow cytometry. The stronger increase in mRNA levels (6-fold) than in cellular fluorescence (3-fold) is not necessarily of biological significance, as it may also result from differences in the sensitivities of the two semiquantitative techniques. The relative fluorescence intensities measured by flow cytometry also suggested that culture at 37 C up-regulates surface expression of MHC class I antigens by isolated ß-cells. The lower levels after culture at 20 C support the view that the lower alloreactivity of such islet cell preparations is related to their lower degree of MHC class I expression (4). The stimulatory action of interferon-{gamma} on MHC class I expression by rat pancreatic cell preparations was not ß-cell specific, as it was also noticed on islet endocrine non-ß-cells and on non-endocrine ductal cells. As in rat islets, interferon-{gamma} also increased mRNA levels and the surface expression of MHC class I antigens of human islet cells. In both rat and human islet cell preparations, elevated glucose levels increased MHC class I mRNA levels 2-fold. It is known that glucose stimulates both insulin and non-insulin protein synthesis by pancreatic ß-cells (34). Its stimulatory effect is achieved by a dose-dependent recruitment of cells into biosynthetic activity as well as by an amplification of the activities in recruited cells (35). Our data thus suggest that a hyperglycemic state can by itself increase MHC class I expression in islet ß-cells; its amplifying effect is additive to that of interferon-{gamma}, resulting, together, in a more than 10-fold stimulation.

Our study confirms that MHC class II antigens are not expressed on the surface of rat or human ß-cells (15, 36, 37). No expression was induced by prolonged glucose activation or by exposure to interferon-{gamma}. MHC class II antigens were not detected on endocrine non-ß cells that were isolated from rat organs. Human islet preparations, however, contained a population of contaminating non-endocrine cells with positive membrane staining for MHC class II antigens, in particular after culture with interferon-{gamma}. It was subsequently shown that human ductal cells, which are identified by their cytokeratin 19 marker, exhibit an increased MHC class II expression upon exposure to interferon-{gamma}. This finding raises the possibility that ductal cells, which are located close to islet cells, actively participate in the local immune reactions that occur in autoimmune diabetes. Close association of MHC class II-positive ductal cells to islet endocrine cells might also be responsible for misinterpretations on antigen expression by ß-cells if no measures are taken to identify the cells at the ultrastructural level. We have also previously demonstrated that such steps are necessary before assuming that insulin-positive cells with MHC class II expression correspond to ß-cells with an aberrant expression of MHC class II antigens (36). Immunocytochemical analysis of unpurified islet cell preparations is certainly adequate for detecting large differences in fluorescence staining in the most frequent cell type, but is less accurate for distinguishing small differences, in particular when they occur in a minority of cells. Particular caution is needed for human islet preparations, which are often contaminated by more than 35% non-endocrine cells (24, 25). The present use of purified ß-cell preparations reduced possible interference by other cell types, and the use of flow cytometry allowed a more sensitive quantitative analysis of MHC class II expression. We have to date been unsuccessful in detecting or reproducing conditions that induce MHC class II expression in ß-cells (14, 17). This observation cannot, however, exclude the possibility that such process occurs in a (pre)diabetic pancreas under the influence of as yet unidentified factors.


   Acknowledgments
 
We thank Nadine Van Slycke for secretarial work, Geert Stangé for technical assistance, the personnel of the Metabolism and Endocrinology and Central Unit ß Cell Transplant (Brussels, Belgium) for preparing rat and human islet cells, and Décio Eizirik for proofreading the manuscript.


   Footnotes
 
1 This work was supported by grants from the Belgian Fonds voor Wetenschappelijk Onderzoek (3.0057.94), the Flemish Community (concerted action 93/019 Matching Fund), and the Juvenile Diabetes Foundation (JDF-DIRP 95–97). This study made use of human islets prepared by the Central Unit of the ß Cell Transplant with financial support from a concerted action in Medical and Health Research of the European Community (BMH-CT95–1561). Back

Received December 3, 1996.

Revised March 3, 1997.

Accepted March 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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